Observations on the interaction between plant growth‐promoting bacteria and the root‐knot nematode Meloidogyne javanica

Abstract Pseudomonas fluorescens, strains L124, L228, L321, and the positive control strain F113 used in this study, produce compounds associated with plant growth promotion, biocontrol, antimicrobial and antiviral activity, and adaptation to stresses. These bacterial strains were tested in vitro and in vivo in tomato plants, to determine their potential role in Meloidogyne javanica suppression. In laboratory experiments, only 2% of M. javanica eggs hatched when exposed to the metabolites of each bacterial strain. Additionally, 100% M. javanica J2 mortality was recorded when nematodes were exposed to the metabolites of F113 and L228. In greenhouse experiments, M. javanica infected tomato plants, which were also inoculated with the bacterial strains F113 and L124, displayed the highest biomass (height, number of leaves, fresh and dry weight) of all bacterial treatments tested. Results from the development and induced systemic resistance experiments indicated that the bacterial strains F113 and L321 had the most effective biocontrol capacity over nematode infection, delayed nematode development (J3/J4, adults and galls), and reduced nematode fecundity. In addition, these results indicated that the bacterial strain L124 is an effective plant growth promoter of tomato plants. Furthermore, it was determined that the bacterial strain L321 was capable of M. javanica biocontrol. P. fluorescens F113 was effective at both increasing tomato plant biomass and M. javanica biocontrol. In an agricultural context, applying successional drenches with these beneficial plant growth promoting rhizobacteria would ensure bacteria viability in the rhizosphere of the plants, encourage positive plant bacterial interactions and increase biocontrol against M. javanica.


| INTRODUCTION
Plant growth-promoting rhizobacteria (PGPR), such as endophytic bacteria, can colonize living plant tissues and maintain a mutualistic relationship with the host plant. There are many benefits involved in such relationships, including increased plant growth and biocontrol against host plant pathogens (Lally et al., 2017;Otieno et al., 2015). Root colonization of these non-pathogenic bacteria can lead to preconditioning plant defenses (Choudhary et al., 2007) by inducing host systemic resistance.
That is, promoting plant health by stimulating their defense mechanisms (Romera et al., 2019) when colonized by beneficial bacterial communities.
Production of secondary metabolites, mainly antibiotics, by PGPR has shown to be an efficient mechanism of nematode control (Cronin et al., 1997;Meyer et al., 2009;Siddiqui & Shaukat, 2003a). The Pseudomonas fluorescens strain F113, used in the present study, is a wellresearched and documented PGPR that was previously proven to be an efficient biocontrol agent against a range of plant pathogenic bacteria, fungi, and nematodes (Redondo-Nieto et al., 2012), including the potato cyst nematode Globodera rostochiensis (Cronin et al., 1997). The antagonistic properties of this rhizobacterium can be attributed to the production of the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG), which is directly linked to the biological control of plant pathogens and the plant parasitic nematodes (PPN) Heterodera glycines, Meloidogyne incognita, and Pratylenchus scribneri (Meyer et al., 2009). Other strains of Pseudomonas are reported to produce the volatile 1H indole-3-carboxaldehyde, which was previously found to have a lethal effect on the bacterial feeding nematode Caenorhabditis elegans (Bommarius et al., 2013). In addition, it has been reported (Kim et al., 2000) that the compound benzenesulfonamide, also produced by some Pseudomonas species, has antifungal properties and can provide biological control of root pathogens.
Root-knot nematodes (RKN) have a large host range and can infect more than 1700 plant species globally, resulting in major crop losses (Pulavarty et al., 2021). These types of RKN, including Meloidogyne javanica, are economically important PPN. They are responsible for significant crop yield losses, in particular with tomato plants, which are among the major vegetable crops grown and consumed worldwide (Perpétuo et al., 2021). In terms of controlling PPN crop infections including from RKN, currently, there are many sustainable practices and strategies employed including crop rotation, growing resistant varieties (Palomares-Rius et al., 2021), and biological control (Pulavarty et al., 2020).
Where possible, often these types of techniques are combined; however, the application of synthetic cheminematicides still dominates. Although these chemicals can be effective, they do not always kill the nematodes in the soil and do not promote sustainability. The use of such chemicals is now heavily regulated due to their hazardous effects on the environment, human health, and lack of efficacy. Plant parasitic nematodes in the soil can become resistant to these chemicals, while nematicide compounds often have nontarget effects. Therefore, the demand for improved, sustainable alternatives is increasing.
Exploiting the potential biocontrol properties of PGPR colonization in plants is a recent development considered for further investigation (Almaghrabi et al., 2013;Jiang et al., 2018;Xiang et al., 2017;Xing et al, 2020). According to dos Santos (2020), there are two systems used by PGPR to promote plant growth. The direct system involves the production of phytohormones and siderophores, nitrogen-fixing and phosphorous solubilization, which are associated with regulating plant growth and development, reproduction, longevity, and plant death (Dilworth et al., 2017;dos Santos et al., 2020 Choudhary et al., 2007) in plants is an enhanced state of defense that can be provoked by the presence of beneficial bacteria, increasing plant protection against subsequent biotic challenges.
This enhanced state of resistance is effective against a broad range of pathogens and parasites (Choudhary et al., 2007). Plant colonization with beneficial bacteria is also effective against the development of giant cells in plant roots, preventing nematode J2 infective juveniles to establish an adequate feeding site (Martinuz et al., 2012) therefore, preventing galls from forming on the roots. Plant root colonization by beneficial bacteria also makes roots less attractive (Martinuz et al., 2012) to infective stage nematodes. If these plant defense mechanisms are triggered by a stimulus such as beneficial bacteria, before plants are infected by plant pathogens, the degree of infection can be reduced (Choudhary et al., 2007).
We hypothesized in this study, that the compounds the bacterial strains were producing would reduce nematode infection and hinder

| Bacterial strains and nematode extraction from roots
Infected roots were placed into a 1 mm sieve, lined with a square of loose weave muslin cloth, and the sieve was placed into a dish of sterile H 2 O for 7 days, ensuring the water was touching the roots until the J2s emerged. On emergence, they were collected on a 40 µm sieve.

| Nematode egg extraction from roots
Infected roots were placed into a screw-topped container and vigorously agitated in 1% sodium hypochlorite solution for 3 min to release the eggs. The suspension was passed through a range of sieves from 150 to 40 µm to trap debris and soil particles in the larger sieves, and the eggs on the smaller sieves. Eggs on the 40 µm sieve were washed with sterile ddH 2 O to remove any sodium hypochlorite (Ganji et al., 2013). At this stage, the eggs were surface sterilized and utilized in the egg hatching experiment in Section 2.2. A deli dish was filled with the sterile egg suspension and incubated for 3-5 days at 28°C (Siddiqui & Shaukat, 2003a) until the sterile J2s were hatched.
These nematodes were used in the ISR experiments in Section 2.3.

| Effects of PGPR metabolites on nematode egg hatching and juvenile mortality
In total, 10 ml of a 24-h bacterial culture (10 8 CFU/ml) grown in nutrient broth (NB) was centrifuged twice at 2800 rpm (965 RCF) for 20 min.
The supernatant was passed through a 0.22 µm sterile filter to remove the bacterial cells and tested for bacterial growth by plating 100 µl on a nutrient agar (NA) culture plate. The assay was established in sterile 24well tissue culture plates, with eight replications of each strain of bacterial metabolites. A suspension, 50 µl in total, containing approximately 100 surface-sterilized nematode eggs, was added to each well holding 450 µl of bacterial supernatant. Untreated controls for the bacterial supernatant were 50 µl of nematode suspension containing surface-sterilized eggs and 450 µl NB. The test was incubated for 5 days at 28°C (Siddiqui & Shaukat, 2003a). The wells were visually inspected for bacterial growth after 5 days of incubation. If growth was evident, the nematodes in that well were not counted. The egg hatching and juvenile mortality were then recorded. Plant roots were stained with acid fuchsin (Bybd et al., 1983) and mounted onto slides. The severity of nematode infection was assessed by counting the number of nematodes at different developmental stages after each harvest, using the descriptions from Dávila-Negrón and Dickson (2013) and Eisenback and Triantaphyllou (2009) (Table 1). In addition, a high-power microscope (Optika) was utilized to take digital images of the developmental stages ( Figure 1), including the presence of galls and egg masses ( Figure 2) using Optika Vision Pro software. As the nematodes were observed in the roots, their stage of development was recorded once for each treatment and the control. Once the nematode developmental stages were identified, the plant roots, stem, and leaves were oven-dried at 60°C and the plant biomass was determined.

| Resistance of treated tomato plants to M. javanica infection
Surface sterilized tomato seeds (Solanum lycopersicum var. "Moneymaker") were planted in pots, 8 cm in diameter, filled with 350 g of sterile soil.
After 3 weeks, the tomato seedlings were uprooted and the roots were split into two halves with a sterile dissecting scalpel. Each half of the root system was immediately transplanted into one pot, 8 cm in diameter and containing 350 g of soil, and the two pots were taped together (see nematode infection, the plants were carefully removed from the pots, the roots were stained by boiling them in 0.1% lactic acid fuchsin (Bybd et al., 1983) and the adult nematodes, galls, and egg masses were identified in the roots and recorded. Overall, statistically significantly (p < 0.001) less than 2% of PPN eggs hatched when exposed to the bacterial metabolite treatments, compared to 73% of those that hatched in the untreated control ( Figure 4). M. javanica juveniles exposed to the bacterial metabolites of L228 and F113 in particular, suffered 100% mortality after 5 days of exposure, compared to 4% mortality in the control treatment, which was statistically significantly lower (p < 0.005). F I G U R E 3 Schematic representation of the establishment of the induced systemic resistance experiment (Siddiqui & Shaukat, 2003a, 2003b. Image 1: Bacterial strains were added to the pots on the left and Meloidogyne javanica J2 was added to the pots on the right; Image 2: No bacteria were added to the pots on the left but J2 was added to the pot on the right.

| Biomass of bacterial inoculated tomato plants infected with M. javanica
The effect of the various treatments, on tomato plant fresh weight ( Figure 6) after 20 days in the presence of M. javanica did not vary much (mean 2.5 g), with the fresh weight of plants treated with F113, L124, and L228 being statistically significantly lower than those in C1 (5 g; p < 0.05). However, a statistically significant increase in tomato plant fresh weight was observed in those treated with F113 (p < 0.05) and after 50 days (18 g), compared to C1 (9 g) and C2 (8 g). In addition, the fresh weight of those treated with L124 (19 g) and F113 (18 g) after 70 days was statistically significantly heavier (p < 0.05) than those in C1 plants (14 g).
On the contrary, the dry weight (Figure 7) of tomato plants after 20 days was highest in those treated with F113 (0.3 g), which was on par with C1 plants (0.3 g). After 50 days of exposure to M. javanica, those plants treated with F113 (1.12 g) and L321 (1.10 g) had the highest dry weight compared to C1 (0.5 g) and C2 (0.9 g). In addition, after 70 days the plants treated with F113 (1.35 g) had the highest dry weight of all treated plants, and C1, also, it was similar in weight to C2 (1.4 g).

| DISCUSSION
The complex tri-trophic interactions among M. javanica, beneficial bacteria, and tomato plants were explored with regard to (1) promoting the growth of M. javanica infected tomato plants and (2) the M. javanica biocontrol capability of the PGPR strains when colonizing infected tomato plants.
In a previous study (Egan, 2019), it has been determined that strain L124 produces compounds associated with biocontrol, strain L228 produces compounds linked with antimicrobial and antiviral activity and strain L321 produces compounds that are associated with adapting to stresses. In addition, P. fluorescens strain F113 produces the secondary metabolite 2,4-DAPG, which has suppressive effects on a wide range of phytopathogens including oomycetes  It is clear from the results in the present study, that the treatments had a negative effect on nematode fecundity, particularly in plants treated with strains L321 and F113. The concept of priming a plant to precondition its defenses before infection (Choudhary et al., 2007) was explored in the ISR experiment in this study, using a In the current study, although the bacterial strains and the nematodes were added to two separate pots, those plants treated with bacterial strains F113 and L321 were indicative of a reduction in plant infection in the pot containing nematodes, compared to the untreated pot ( Figure 9). This result suggests that a systemic resistance response was induced, due to the presence of the bacterial strains on one side of the root system, which subsequently reduced infection in the roots that were exposed to the nematodes, even though they were physically located in a different pot and away from the bacteria. Similarly, Siddiqui and Shaukat (2003a)  The objective of this study was to explore the interaction between M. javanica infected tomato plants and bacteria and to further investigate the bacterial strains' suppressive effects against M. javanica and their plant growth promotion capacity. From these results, it can be recommended that there should be an emphasis on PGPR inoculation of tomato plants as early as possible for the induction of plant defense. In an agricultural context, pretreating seeds before sowing or inoculating seedlings early, before planting in the field, may be an effective approach to the management of PPNs. There is a concern, however, on the viability of the bacterial strains, in terms of their capacity to colonize host plants and the extent they would remain within the plant, to induce a systemic resistance response in a field situation (Ji et al., 2019). Application rates of the suppressive bacteria and nematode population density, in the soil and plants, play an important role in the degree of suppression of PPNs (Siddiqui & Shaukat, 2003a). Moreover, it is known that the production of antibiotics by beneficial bacterial strains, can improve their ecological fitness (Chandra & Kumar, 2017), which can further influence long-term antagonistic effects against nematodes. Therefore, it is recommended that, along with pre-treating seeds (Xing et al., 2020)

| CONCLUSION
The results reported here suggest that some bacterial strains used in this study, and the components they produce (1) affect the capacity of M.
javanica to infect their host and (2)